Bacteria constitute the majority of the world's biomass and are responsible for most bioconversion on the planet. Bacterial pathogens, on the other hand, present major threats to human health, causing numerous infectious diseases in humans. Moreover, bacteria also serve as model organisms for us to understand fundamental biological processes, especially at the molecular and cellular levels. It is thus of paramount importance to understand how molecules coordinate and interact inside bacterial cells to support life processes. In bacteria, life processes take place in a small volume of <1<m3, where the chromosome and thousands of different proteins, RNA, and small molecules reside. It is now recognized that a bacterial cell is not simply a bag of enzymes, but a highly organized and orchestrated system. However, the small sizes of these cells have made it extremely difficult to probe their sub-cellular organization, due to the lack of tools with sufficient spatial and temporal resolution required to elucidate the structure and dynamics of molecular assemblies within such a small volume. Our understanding of bacterial cellular organization is thus still primitive, lagging far behind that of eukaryotic cells. For most bacterial proteins, we do not know their quantities inside the cell, nor do we know their spatial distributions and ultra-structural organization, let alone their in vivo dynamics. This knowledge deficit has severely hampered our understanding of how bacteria function. Thanks to recent developments in single-molecule detection, super-resolution imaging, and the construction of a fluorescent E. coli library in the PIs' labs, we are now in a position to have the first high-resolution, integral view of live bacteria. By combining these bioimaging and systems biology tools, we propose to quantify the entire E. coli proteome with single-molecule sensitivity, to map the intracellular distributions and ultra-structural organization of most E. coli proteins with nanometer resolution, and to follow their dynamic changes and interactions in real time in living cells. Based on such knowledge, we plan to construct a quantitative, high- resolution map of E. coli cellular architecture with the molecular specificity of each individual gene. Furthermore, we plan to profile changes of this architecture in response to cellular states and environmental conditions. This unprecedented, system-wide view of bacterial cellular architecture with ultimate sensitivity and resolution will not only address a wide range of questions in bacteriology, but will also have a broad impact on microbiology and biomedical research.